INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : III. Tubular Gland Cell Cytodifferentiation

Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells synthesize the major egg-white protein, ovalbumin. Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation. Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days. Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized. Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment. When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1/20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment. Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone. Instead, these cells appear to transform into other cell types—some with cilia and some with unusual flocculent granules. We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists. P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : I. Antagonistic Effect of Progesterone on Estrogen-Induced Proliferation and Differentiation of Tubular Gland Cells

Daily administration of estrogen to immature female chicks results in marked oviduct growth and appearance of characteristic tubular gland cells which contain lysozyme. Although a rapid increase in total DNA and RNA content begins within 24 hr, cell specific protein, lysozyme, is first detectable after 3 days of estrogen. Progesterone administered concomitantly with estrogen antagonizes the estrogen-induced tissue growth as well as appearance of tubular gland cells and their specific products, lysozyme and ovalbumin. When the initiation of progesterone administration is delayed for progressively longer periods (days) during estrogen treatment, proportionally greater growth occurs with more lysozyme and tubular gland cells after 5 days of total treatment. Progesterone does not inhibit the estrogen-stimulated increase in uptake of α-aminoisobutyric acid and water by oviduct occurring within 24 hr or the estrogen-induced increase in total lipid, phospholipid, and phosphoprotein content of serum. The above results of progesterone antagonism can best be explained by the hypothesis that progesterone inhibits the initial proliferation of cells which become tubular gland cells but does not antagonize the subsequent cytodifferentiation leading to the synthesis of lysozyme and ovalbumin once such cell proliferation has occurred.     

Commitment of chick oviduct tubular gland cells to produce ovalbumin mRNA during hormonal withdrawal and restimulation

Acute withdrawal of estrogen from chicks leads to a precipitous decline in egg white protein synthesis and egg white mRNAs in the oviduct. In this paper we explore the biochemical basis of this phenomenon as well as the capacity of the "withdrawn" tubular gland cells to be restimulated with steroid hormones. During withdrawal, the decline in ovalbumin mRNA was closely correlated with the decline in nuclear estrogen receptors. Within 2-3 d of estrogen removal a withdrawn state was established and then maintained, as defined by a 1,000-fold-lower level of ovalbumin mRNA and a 20-fold-lower level of nuclear estrogen receptors, relative to the estrogen-stimulated state. The number of active forms I and II RNA polymerases declined by 50% during this time. Histological examination of oviduct sections and cell suspensions, combined with measurements of DNA content, revealed that tubular gland cells persisted as a constant proportion of the cell population for 3 d after estrogen removal. Despite a 1,000-fold decrease in the content of ovalbumin mRNA, the ovalbumin gene remained preferentially sensitive to digestion by DNase I. When 3-d-withdrawn oviducts were restimulated with either estrogen or progesterone, in situ hybridization revealed that greater than or equal to 98% of the tubular gland cells contained ovalbumin mRNA. Induction by a suboptimal concentration of estrogen was correlated with a lower concentration of ovalbumin mRNA in all cells rather than fewer responsive cells.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : II. Effects of Estrogen and Progesterone on Tubular Gland Cell Function

The effects of estrogen and progesterone on the function of chick oviduct tubular gland cells have been studied. Such function, as measured by the increase in specific cell products such as lysozyme and ovalbumin, requires the continuous presence of estrogen or progesterone. Withdrawal of hormone results in a rapid cessation of function and an involution of the oviduct accompanied by rapid decreases in total weight, lysozyme, and RNA. During such involution, tubular gland cells per se persist, as evidenced by a lack of comparable decrease in total DNA content and by histological demonstration of tubular gland cells. When estrogen administration is reinstituted, preexisting tubular gland cells rapidly synthesize ovalbumin and lysozyme without requiring new DNA synthesis. Administration of progesterone also stimulates the function of such cells. Furthermore, the effects of estrogen and progesterone are synergistic on the synthesis of lysozyme and ovalbumin, whereas progesterone antagonizes the estrogen-evoked formation of tubular gland cells. It is suggested that such complex interactions of estrogen and progesterone on oviduct development and function result from differences in responsiveness of the various cell types present in the tissue.     

In situ hybridization of ovalbumin mRNA in the chick oviduct reveals target cell specificity for estrogen and progesterone

An in situ hybridization method using paraffin-embedded sections was used to characterize the chicken oviduct cells synthesizing ovalbumin mRNA due to the action of estrogen and progesterne. The cytodifferentiation of the oviduct cells was induced by 17β-estradiol administration to newly hatched female chicks. To avoid possible effect of estrogen on the action of progesterone the chicks were withdrawn from the estrogen by six days withdrawal period without hormone treatment. Ovalbumin mRNA was not synthesized after a period of estrogen withdrawal. Administration of estrogen induced ovalbumin mRNA in the tubular gland cells. Administration of progesterone induced the expression of ovalbumin mRNA in the surface epithelial cells. It was also found that progesterone induced mucus producing goblet cells in the surface epithelium. Estrogen did not have an effect on the mucus production, which suggests that progesterone could induce the terminal differentiation of the goblet cells. We conclude that the expression of ovalbumin in the surface epithelial cells and in the tubular gland cells is specific for progesterone and estrogen, respectively.     

Down regulation of c-myc, c-fos and erb-B during estrogen induced proliferation of the chick oviduct

Oncogenes c-myc, H-ras, c-fos and erb-B were constitutively expressed in immature chick oviduct withdrawn from estrogen administration for 2.5 weeks after 10 d of primary estrogen stimulation. Following secondary estrogen stimulation of the withdrawn chicks, synthesis of egg white proteins is rapidly induced and remaining non-functioning tubular gland cells are stimulated to proliferate with a doubling time of 24 h. During first 12 h of secondary estrogen stimulation, H-ras mRNA levels doubled and did not increase further at 24 h and 48 h. The steady state levels of c-myc, erb-B and c-fos mRNA decreased 24 h following secondary estrogen stimulation. The levels of these oncogene RNAs in oviduct were similar at 48 h following secondary estrogen stimulation to those from immature chicks administered 10 d of primary estrogen stimulation. Thus elevated expression of c-myc and c-fos mRNA does not appear to be necessary components for sustained estrogen induced cell proliferation in the chick oviduct.     

Expression of the avian c-erb B (EGF receptor) protooncogene during estrogen-promoted oviduct growth

Expression of cellular erb B protooncogene messenger RNAs has been analyzed in the oviducts of immature chicks during estrogen-promoted growth. Hybridization of oviduct total cellular RNA with viral-derived erb B oncogene probes demonstrated significant expression of c-erb B mRNA in oviduct cells of untreated chicks. Daily administration of estrogen (diethylstilbestrol) to chicks results in marked oviduct growth but did not appreciably affect expression levels of c-erb B messenger RNA in oviducts after 2, 4 or 6 days of treatment. Withdrawal of chicks from estrogen treatment resulted in termination of oviduct growth. However, c-erb B messenger RNAs were detectable in the nonproliferative tissue at 5 days after hormone withdrawal. Readministration of diethylstilbestrol, progesterone or diethylstilbestrol plus progesterone to hormone-withdrawn birds (secondary stimulation) also did not affect c-erb B messenger RNA levels in the oviduct. These results demonstrate significant expression of the cellular erb B (epidermal growth factor receptor) gene in the avian oviduct. However, EGF receptor messenger RNA synthesis is not modulated in the oviduct by steroid hormones.     

Studies on sex-organ development. Prenatal effect of oestrogenic hormone on tubular-gland cell morphogenesis and ovalbumin-gene expression in the chick Müllerian duct.

The effects of diethylstilboestrol on morphogenesis and cyto-differentiation of the chick-embryo left Müllerian duct were examined. Embryos were treated at different stages of development with maximal-responsive doses of diethylstilboestrol over a 5-day interval. The shell gland and magnum regions of the Müllerian duct were then assayed for growth and histological morphogenesis. The results were correlated with diethylstilboestrol-induced ovalbumin-gene expression as measured by ovalbumin-mRNA (mRNAov) accumulation and the relative rate of ovalbumin synthesis. Treatment of the embryo from day 10 to day 15 of incubation induces morphogenesis of tubular-gland cells in the Müllerian-duct magnum. Although these cells constitute 10% of the total cell population and contain an average of 8000 molecules of mRNAov per cell, ovalbumin synthesis is only 0.85% of total magnum protein synthesis. The Müllerian-duct magnum of embryos treated from day 13 to day 18 of incubation contains about 30% tubular-gland cells, which have accumulated an average of 7000 molecules of mRNAov per cell, but ovalbumin synthesis is only 3.25% of total magnum protein synthesis. The Müllerian-duct magnum of embryos treated from day 16 to day 21 of incubation contains about 50% tubular-gland cells, which have accumulated an average of 6500 mRNAov molecules per cell, and ovalbumin synthesis is 10% of total magnum protein synthesis. Oestrogen responsiveness develops simultaneously in the Müllerian-duct magnum and shell-gland regions. Compared with the rate of diethylstilboestrol-induced oviduct growth, the relative rate of diethylstilboestrol-induced Müllerian-duct growth increases with embryonic age, from 20-fold lower in the 10-day embryo to only 3-fold lower in the 16-day embryo. All results are discussed in comparison with the responses to oestrogen of the immature chick oviduct, and in terms of the ontogeny of hormone-competent epithelial and stromal components of the Müllerian duct. It is concluded that the development of oestrogenic competence in the embryonic Müllerian duct is a multiphasic phenomenon. A dramatic increase in hormone responsiveness in the Müllerian duct occurs between days 10 and 16 of development, and a less dramatic final maturation of oestrogen responsiveness occurs between day 16 of development and 1 week after hatching.     

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine

A Pseudo-ovalbumin gene, bearing significant nucleotide sequence homology to the ovalbumin gene, has been cloned from genomic chick DNA. Similar to the authentic ovalbumin gene, the pseudo-gene is a unique sequence gene in the chick genome and is expressed at a low level in the immature chick oviduct. In contrast to the ovalbumin gene, expression of the pseudo-gene in the oviduct is not inducible by estrogen. The concentration of pseudo-gene RNA is only ～0.01% of that of authentic ovalbumin mRNA in estrogen-stimulated oviduct cells. Nucleotide sequence analysis of the two sequence related genes may reveal the molecular basis of differential response to steroid hormone induction in the same tissue.     

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.     

Isolation of a nuclear ribonucleoprotein fraction from chick oviduct containing ovalbumin messenger RNA sequences

A method was developed for the isolation of a ribonucleoprotein fraction from chick oviduct nuclei that contains 70% of the pulse-labeled RNA. These fractions also contain about 1% of the nuclear DNA and have an average RNA to DNA ratio of about 4:1. The major nuclear RNP proteins of 32,000 M_r are present along with many additional proteins including histories. However, polysomal proteins and major oviduct cytoplasmic proteins are absent. Nuclei from fully stimulated chick oviduct contain about 3000 copies of ovalbumin messenger RNA sequences of which about 200 are in the RNP complexes: these complexes have sedimentation coefficients of 30 to 350 S and are resistant to disruption by EDTA.The level of ovalbumin mRNA sequences in these complexes reflects the overall rate of synthesis of this RNA. Withdrawal of estrogen leads to a parallel decline of nuclear estrogen receptors and ovalbumin mRNA sequences in the RNP complexes and a subsequent loss of cytoplasmic ovalbumin mRNA about three hours later. The 300-fold decrease in the level of ovalbumin mRNA sequences in these complexes and the eightfold decrease in stability of cytoplasmic ovalbumin mRNA account for the 2500-fold decrease in the level of cytoplasmic ovalbumin mRNA observed during withdrawal. Upon stimulation with estrogen, the kinetics of reappearance of ovalbumin mRNA sequences in the RNP complexes apparently accounts for the accumulation of cytoplasmic ovalbumin mRNA. Thus the nuclear RNP has some of the properties expected of nascent RNP complexes.The levels of ovalbumin and conalbumin mRNA sequences increase in the nuclear RNP with markedly different kinetics: conalbumin mRNA sequences reach half maximum by 1.5 hours, whereas ovalbumin mRNA sequences in these complexes reach half maximum at about eight hours. In the analysis in the accompanying Appendix, we show that the immediate increase of conalbumin mRNA sequences in the nuclear RNP may be accounted for by interaction of the hormone receptor complex with a single regulatory site, whereas the delayed increase of ovalbumin mRNA sequences in the RNP may be due to a requirement for interaction of the hormone receptor complex with multiple regulatory sites.     

Estrogen Receptor Is Not Primarily Responsible for Altered Responsiveness of Ovalbumin mRNA Induction in the Oviduct From Genetically Selected High- and Low-Albumen Chicken Lines

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1,the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.     

The effect of dihydrotestosterone on the estrogen-induced cytodifferentiation of the chick oviduct

The androgenic effects on the estrogen-induced cytodifferentiation of the chick oviduct epithelium were investigated. Dihydrotestosterone was shown to have an effect on the organization of stromal cells. Since these cells contained androgen receptor (AR), it is reasonable to assume an involvement of androgens in the differention and functioning of these cells through a direct action. Immunohistochemical analysis revealed a wide distribution of AR. AR was shown to be expressed in both the endothelial and smooth musle cells of blood vessels. In the immature oviduct AR was located in the epithelial, mesenchymal and mesothelial cells. In the differentiating oviduct, whether induced by exogenous estrogen or normally by endogenous hormones, AR was also expressed by the tubular gland cells. Dihydrotestosterone alone had no effect on the morphology of the immature oviduct, suggesting the involvement of the determinants of differentiation in the action of androgen together with estrogen.